ABSTRACT: All three projects in this program require large amounts of recombinant Hb mutants to evaluate the causes of acellular Hb toxicity in simple cellular systems, organs, and whole animals. The work with perfused hearts and whole animal exchange transfusion models requires scaling up our current production of -0.5 to 1 g of rHb per week by at least 10-fold. We also need to achieve complete removal of endotoxin (LPS), which requires dedicated purification facilities to remove all traces of E. coli proteins and membranous materials from final preparations and special packaging. These goals will be achieved by setting up a rHb prodUctioh facility at Rice University, which will bper'ate cohtihuously to pr'bvide the LPS- free rHb solutions for Projects 1, 2, and 3. A full time Ph.D. staff scientist will run the facility with the help of a research assistant and other members of the Pi's laboratory, who are part of Project 2. A 30 L B.Braun Biostat C+ 30 L will be purchased and dedicated solely to rHb production and be run in parallel with our existing 20L Biostat C fermentor, whenever possible. Our specific aims are to: (1) produce at least 6 g of LPS-free recombinant Hb samples per week or ^ 300 grams per year using current expression and production technology; and (2) enhance these production yields 2 to 3-fold by integrating the Plesiomonas shigelloides heme transport system (heme utilization genes, hug) and alpha hemoglobin stabilizing protein (AHSP) into the expression protocol to facilitate holoprotein assembly and to increase the efficiency of purification by decreasing protein miss-folding and preventing the accumulation of hemin degradation products during bacterial growth.